STUDIES ON THE ENCYSTATION OF ENTAMOEBA

以入侵内阿米巴的无菌包囊体系为包囊模型，研究了核复制DNA聚合酶的特异性抑制剂阿米迪林对包囊的影响。Aphidicolin对包囊有抑制作用，其作用是可逆的。当滋养体用药物预处理时，包囊几乎完全被抑制。这可能是由于腺苷二磷胆碱处理使处于G1/S交界处的细胞聚集，在囊化培养液中既不能合成脱氧核糖核酸，也不能分裂，而未经处理的细胞包括已经合成脱氧核糖核酸的G2和M细胞，因此不受腺苷二磷胆碱的影响。与滋养体相比，包囊中DNA聚合酶活性水平随着包囊的进行而降低。结果表明，包囊伴随dna聚合酶活性水平的降低，这与先前发现的核分裂发生在…上是相关的。在缺乏DNA合成的情况下，更多的环状包囊成熟。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和免疫印迹法检测了侵袭性肠杆菌包囊中包囊特异性蛋白的出现及其免疫原性。兔抗包囊抗体可与滋养体蛋白和包囊蛋白反应。最有趣的结果是，兔抗囊血清在孵育1天后主要与88 kDa的包囊蛋白反应。88 kDa蛋白与被滋养体蛋白吸收的抗囊血清反应，因此是包囊特异的，该蛋白存在于富含细胞壁碎片的颗粒部分中。结果表明，包囊伴随着包囊特异的88 kDa糖蛋白的出现，这种蛋白是免疫显性的，孵化1天后在包囊中表达最丰富，似乎与囊壁有关。研究了微丝特异性抑制剂细胞松弛素D对侵袭性肠杆菌包囊的影响。细胞松弛素D以剂量依赖的方式抑制囊化作用。这是微丝参与囊化的第一个证据。在细胞松弛素D作用下生长的滋养体转移到包囊剂中后形成多核，并产生多核包囊。药物对滋养体细胞松弛素D的抑制作用强于未用药的滋养体。因此，细胞松弛素D使滋养体多核对包囊有抑制作用。较少

Using an axenic encystation system of Entamoeba invadens as a model for E. histolytica encystation, effect of aphidicolin, a specific inhibitor of nuclear replicative DNA polymerases, on the encystation was examined. Aphidicolin inhibited the encystation and its effect was reversible. Encystation was almost completely inhibited when trophozoites were pretreated with the drug. This can be explained that treatment with aphidicolin accumulates the cells at G1/S border and those cells can neither synthesize DNA nor divide when placed in encystation medium, whereas the cells without the pretreatment include G2 and M cells which have already synthesized DNA so that they are not affected by aphidicolin. The level of DNA polymerase activity in cysts decreased as encystation proceeded as compared with that of trophozoites. The results indicate that encystation is accompanied by a reduced level of DNA polymerase activity, which correlates with the previous finding that nuclear division occurs du … More ring cyst maturation in the absence of DNA synthesis. The appearance of cyst-specific proteins in encysting E. invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. A rabbit antiserum against cysts reacted with the trophozoite proteins as well as cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific and the protein was found to be present in the particulate fraction, which was rich in cell-wall fragments. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall. Effect of cytochalasin D, a specific inhibitor of microfilaments, on the encystation of E. invadens was examined. Encystation was inhibited by cytochalasin D in a dose-dependent manner. This is the first evidence for participation of microfilaments in the encystation. Trophozoites grown with cytochalasin D became multinucleate and produced multinucleate cysts when transferred to encystation medium. Encystation with cytochalasin D of trophozoites grown with the drug was more inhibited than that of trophozoites grown without the drug. Thus multinucleation of trophozoites by cytochalasin D had an inhibitory effect on the encystation. Less